ABSTRACT

The present study was conducted to monitor wild birds based on the concern that they could disseminate avian influenza virus (AIV) between Mongolia and Korea, which shares the same migratory flyway. Of 1,528 fecal samples analyzed, 21 low-pathogenic AIV were isolated from 2007 to 2009. Nineteen AIV-positive fecal samples were identified as Anseriformes by DNA bar coding. The most frequently isolated subtype was H3 (61.9%), and the most prevalent hemagglutinin/neuraminidase combination was H3N8 (52.4%). Phylogenetic analysis was performed to assess their genetic relationships with those of domestic poultry and wild birds in Korea. The H3 and H7 surface genes belonged to the Eurasian lineage and clustered together in a group with Korean wild birds and poultry. Most N8 genes clustered phylogenetically with viruses isolated in Eurasia, whereas 1 of the Mongolian viruses and some Korean viruses belonged to the North American lineage. The polymerase acidic protein of the internal gene was not distinguishable from the H5N1 highly pathogenic AIV of the goose/Guangdong/1/1996 (Gs/Gd)-like virus. Our study suggests that Mongolian AIV isolates have evolved with genetically multiple genotypes and are closely related to those of AIV in poultry as well as in wild birds in Korea.

INTRODUCTION

Influenza A virus is a negative-sense, single-stranded RNA virus that has 8 gene segments coding for 11 proteins, in which 2 integral surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA), harbor 16 and 9 serotypes, respectively (Fouchier et al., 2005). Because wild waterfowl play a vital epidemiological role in the emergence of influenza viruses that are potentially hazardous for humans and animals, concern regarding the surveillance of avian influenza viruses (AIV) in these vast natural reservoirs has increased recently around the globe (Jahangir et al., 2010).

Geographically, large and small wetlands located in the north-central region of Mongolia have been coincident with sites of H5N1 highly pathogenic avian influenza (HPAI) outbreaks since 2005 (Figure 1). These habitats are congregation and breeding sites for many migratory birds from 2 main flyways: the East Asian-Australasian flyway and the Central Asian flyway (Newton, 2008). Korea shares the East Asian-Australasian flyway with Mongolia and harbors a large poultry population on farms, at residences (i.e., backyard facilities), and in live bird markets (Song et al., 2008; Lee et al., 2010b; Kang et al., 2011). Live bird markets in particular may act as a link between the wild host and intensively reared poultry and may extend the genetic diversity of AIV in Korea (Song et al., 2008; Kang et al., 2009; Lee et al., 2010b).

Figure 1

Location of the sampling sites and outbreak sites of H5N1 highly pathogenic avian influenza (HPAI) in Mongolia. Black circles represent the sampling sites during this study, and empty circles denote the outbreak sites of H5N1 HPAI in wild birds in Mongolia, 2005 to 2010. Color version available in the online PDF.

Figure 1

Location of the sampling sites and outbreak sites of H5N1 highly pathogenic avian influenza (HPAI) in Mongolia. Black circles represent the sampling sites during this study, and empty circles denote the outbreak sites of H5N1 HPAI in wild birds in Mongolia, 2005 to 2010. Color version available in the online PDF.

Four outbreaks of H5N1 HPAI have occurred in poultry and wild birds in Korea, which have exacted serious loss of life and economic loss on chickens and duck farms (Lee et al., 2005, 2008; Kim et al., 2010). In particular, the second outbreak in 2006/2007 and the fourth outbreak, which began in 2010 and which is still ongoing, have been ominously notable given the subsequent emergence of clade 2.2 and clade 2.3.2 H5N1 virus, respectively, in migratory birds in northeastern Asia, including Mongolia, because it highlights the possible role of migrating wild birds in the dissemination of H5N1 to domestic poultry.

We presume that AIV isolated from wild birds and poultry between Mongolia and Korea have epidemiological relationships. Therefore, the main objectives of this study were to survey AIV circulating in wild birds in Mongolia and to analyze their genetic characterization and relationships with Korean isolates from domestic poultry and poultry from live bird markets as well as from wild birds.

MATERIALS AND METHODS

Sample Collection

Fresh fecal samples from wild birds were collected from May to October to survey for AIV in Mongolia between 2007 and 2009. Fecal samples were collected after observing defecation. The sample collection sites included major wild bird habitats and outbreak sites of H5N1 HPAI in wild birds in Mongolia from 2005 to 2010 (Figure 1). Table 1 displays the numbers of samples and isolation of influenza A viruses during the survey period. All samples were transported from Mongolia to the diagnostic laboratory of the National Veterinary Research and Quarantine Service in Korea in a refrigerated container and were stored at 4°C until assayed.

Numbers of samples and virus isolation

Table 1
Numbers of samples and virus isolation
Year Total samples
(feces) 
Virus isolation 
No. of
positive 
% of
positive 
2007 340 17 5.0 
2008 963 0.42 
2009 225 
Total 1,528 21 1.37 
Year Total samples
(feces) 
Virus isolation 
No. of
positive 
% of
positive 
2007 340 17 5.0 
2008 963 0.42 
2009 225 
Total 1,528 21 1.37 

Virus Isolation and Identification

Each sample was suspended in an antibiotic solution and centrifuged at 2,300 × g for 15 min at 4°C. Each supernatant was inoculated into the allantoic cavity of 10-d-old embryonated hen’s eggs and incubated at 37°C for 4 to 5 d. Allantoic fluid from the incubated eggs was harvested and centrifuged for purification. The presence of virus was determined by an HA assay, and detected viruses were subtyped by reverse transcription-PCR using influenza-specific primers (Lee et al., 2001) and by an NA inhibition test (World Health Organization Expert Committee, 1980).

Bird Species

A bar-coding system using mitochondrial DNA of bird feces (Hebert et al., 2004; Lee et al., 2010a) was used to determine host species in this study. Mitochondrial DNA was extracted from fresh fecal samples by using a DNA Stool Mini Kit (Qiagen, Valencia, CA) according to the manufacturer’s protocol. Universal and modified primers were used to amplify the gene encoding mitochondrial cytochrome oxidase gene subunit I present in host feces (Lee et al., 2010a). The generated PCR products were sequenced and identified using information contained on the Barcode of Life Data Systems web site (http://www.boldsystems.org/views/login.php).

Molecular and Phylogenetic Analyses

Viral genes were sequenced and analyzed as described previously (Hoffmann et al., 2001; Kang et al., 2011). Briefly, viral RNA was extracted from the allantoic fluid of embryonated eggs by using a Viral Gene-Spin Viral DNA/RNA Extraction Kit (iNtRON, Seongnam, South Korea). Segments of the genes were amplified with gene-specific universal primers (Hoffmann et al., 2001) and were newly designed as necessary. Polymerase chain reaction products were purified from agarose gels by using a Qiaquick Gel Extraction Kit (Qiagen), and sequencing of the PCR product was performed at Macrogen (Seoul, South Korea) with an ABI 3730 XL DNA sequencer (Applied Biosystems). The nucleotide sequences of the Mongolian isolates obtained in this study were deposited in the GenBank database system (accession no. JN029540-JN029689; http://www.ncbi.nlm.nih.gov/nuccore).

Assembly of the sequencing contigs and translation of the collated nucleotide sequences into a deduced amino acid sequence were performed using the VectorNTI Advance program (Invitrogen, Carlsbad, CA). The sequence data were aligned using the AlignX multiple sequence alignment in the VectorNTI Advance program (Lu and Moriyama, 2004). A phylogenetic tree was constructed using the neighbor-joining method within Clustal X version 1.83 (KVL Bioinformatics, Copenhagen, Denmark), with 1,000 bootstrapping replicates. The final tree outfile was visualized using TreeView Win32 software (http://taxonomy.zoology.gla.ac.uk/rod/treeview.html). For phylogenetic analysis, we used the surface genes and internal genes of all Mongolian isolates obtained in this study. Other available sequences used for genetic comparison were obtained from the National Center for Biotechnology Information database (http://www.ncbi.nlm.nih.gov/).

RESULTS

Isolation of AIV from Wild Birds in Mongolia

For AIV surveillance, 1,528 fecal samples were collected in northern and southeastern Mongolia from 2007 to 2009 (Table 1 and Figure 1). Three hundred forty samples were collected in 2007, of which 17 (5.0%) were positive. Subtypes were H3N8 (n = 8), H4N6 (n = 4), H7N7 (n = 1), H4N2 (n = 1), H3N1 (n = 1), H3N2 (n = 1), and H10N6 (n = 1; Table 2). A total of 963 samples were collected in 2008, 4 of which (0.42%) were AIV positive (H3N8, n = 3; H7N9, n = 1). None of the 225 samples collected in 2009 contained AIV. The total isolation rate was 1.37%.

Avian influenza virus (AIV) isolates in wild birds in Mongolia from 2007 to 2008

Table 2
Avian influenza virus (AIV) isolates in wild birds in Mongolia from 2007 to 2008
Year  Region (lake)  Species1  Subtype AIV
isolate 
2007  Bulgan (Khunt)  Unidentified  1 × H7N7 (LPAI2
   Sukhbaatar (Ganga)  Whooper swan (Cygnus cygnus 5 × H3N8, 1 × H3N1 
      Ruddy shelduck (Tadorna ferruginea 1 × H3N8 
      Unidentified  1 × H4N6 
   Sukhbaatar (Holbo)  Pintail (Anas acuta 2 × H4N6, 1 × H3N8, 1 × H4N2 
      Ruddy shelduck (Tadorna ferruginea 1 × H3N8, 1 × H3N2 
      Northern lapwing (Vanellus vanellus 1 × H4N6 
      Canvasback (Anthya valisineria 1 × H10N6 
2008  Bulgan (Khunt)  Snow goose (Anser caerulescens 2 × H3N8 
   Arkhangai (Ugii)  White-fronted goose (Anser albifrons 1 × H3N8 
   Uvs (Uvs)  Wild duck3  1 × H7N9 (LPAI) 
Year  Region (lake)  Species1  Subtype AIV
isolate 
2007  Bulgan (Khunt)  Unidentified  1 × H7N7 (LPAI2
   Sukhbaatar (Ganga)  Whooper swan (Cygnus cygnus 5 × H3N8, 1 × H3N1 
      Ruddy shelduck (Tadorna ferruginea 1 × H3N8 
      Unidentified  1 × H4N6 
   Sukhbaatar (Holbo)  Pintail (Anas acuta 2 × H4N6, 1 × H3N8, 1 × H4N2 
      Ruddy shelduck (Tadorna ferruginea 1 × H3N8, 1 × H3N2 
      Northern lapwing (Vanellus vanellus 1 × H4N6 
      Canvasback (Anthya valisineria 1 × H10N6 
2008  Bulgan (Khunt)  Snow goose (Anser caerulescens 2 × H3N8 
   Arkhangai (Ugii)  White-fronted goose (Anser albifrons 1 × H3N8 
   Uvs (Uvs)  Wild duck3  1 × H7N9 (LPAI) 

1Bird species were identified by a DNA bar-coding system, which uses mitochondrial DNA of bird feces to determine the host species.

2LPAI = low-pathogenic avian influenza.

3Wild duck indicates a mallard or spot-billed duck. The bar-coding system cannot differentiate between a mallard and spot-billed duck.

Concerning the spatial distribution of the isolation rates, the prevalence of AIV was highest in southeastern Mongolia, at Sukhbaatar (16 isolates), which was also the site of the May 2010 outbreak of H5N1 HPAI (Table 2 and Figure 1). Four AIV were isolated from Bulgan and Arkhangay, which were the outbreak sites of H5N1 HPAI in 2005/2006 and 2009, respectively, and 1 H7 virus was isolated from northwestern Mongolia, at Uvs. There was no isolation at Khuvsgel, the site of a 2005 outbreak involving H5N1 HPAI.

Of the 21 AIV, HA subtypes H3, H4, H7, and H10 and NA subtypes N1, N2, and N6 to N9 were detected. The H3 (61.9%) and H4 (23.8%) were the most abundantly detected HA subtypes, followed by H7 (9.5%) and H10 (4.8%). The most frequently detected NA subtypes were N8 (52.4%), followed by N6 (23.8%), N2 (9.5%), and 1 each (4.8%) of N1, N7, and N9. In total, 8 different HA/NA subtype combinations were detected. The most frequently detected subtype combinations were H3N8 and H4N6, which comprised 52.4 and 19.0% of all isolated influenza A viruses, respectively. Each of the H3N1, H3N2, H4N2, H7N7, H7N9, and H10N6 subtype combinations was detected (Table 2).

Prevalence of Bird Species in AIV-Positive Samples

The DNA bar-coding system for the identification of host species in AIV-positive fecal samples was incorporated into the analyses (Table 2). Among 21 AIV-positive fecal samples, 19 AIV were isolated from 6 different bird species, which were identified as Anseriformes: Whooper swan (Cygnus cygnus, n = 6, 28.6%), pintail (Anas acuta, n = 4, 19%), ruddy shelduck (Tadorna ferruginea, n = 3, 14.3%), snow goose (Anser caerulescens, n = 2, 9.5%), northern lapwing (Vanellus vanellus, n = 1, 4.8%), canvasback (Aythya valisineria, n = 1, 4.8%), white-fronted goose (Anser albifrons, n = 1, 4.8%), and wild duck (Anas platyrhynchos or Anas poecilorhyncha, n = 1, 4.8%). The hosts of the 2 remaining AIV-positive samples were not identified.

Phylogenetic Analysis of the Surface Genes

Phylogenetic analysis for the surface genes of Mongolian isolates was performed to assess their genetic relationships with those of domestic poultry and wild birds in Korea (Figure 2a to 2c). The HA genes of 13 H3 viruses isolated from Mongolian wild birds in 2007 to 2008 clustered in the Eurasian avian lineage and showed genetic diversity within the Eurasian lineage; these were similar to the viruses isolated from Korean wild birds. However, the isolates of Korean poultry had an independent lineage distinguishable from the isolates in wild birds from both countries. The Mongolian isolates showed 93.6 to 99.5% homology with themselves and 88.4 to 97.8% and 91.3 to 92.5% homology with those of wild birds and poultry in Korea, respectively (Figure 2a).

Figure 2

Phylogenetic trees of the surface genes: (a) H3, (b) H7, and (c) N8. The Mongolian viruses isolated are denoted in boldface, and Korean viruses isolated in poultry and wild birds or available in GenBank (http://www.ncbi.nlm.nih.gov/genbank/) are italicized. The nucleotide sequences were analyzed using Clustal X (version 1.83; KVL Bioinformatics, Copenhagen, Denmark), and phylogenetic trees were constructed by the neighbor-joining method. The robustness of groupings was assessed by bootstrap resampling of 1,000 replicate trees.

Figure 2

Phylogenetic trees of the surface genes: (a) H3, (b) H7, and (c) N8. The Mongolian viruses isolated are denoted in boldface, and Korean viruses isolated in poultry and wild birds or available in GenBank (http://www.ncbi.nlm.nih.gov/genbank/) are italicized. The nucleotide sequences were analyzed using Clustal X (version 1.83; KVL Bioinformatics, Copenhagen, Denmark), and phylogenetic trees were constructed by the neighbor-joining method. The robustness of groupings was assessed by bootstrap resampling of 1,000 replicate trees.

Genetic analysis for the HA genes of the 2 Mongolian H7 viruses showed that the protease cleavage site had the typical motif of a low-pathogenic H7 (PEIPKGR/GLF) virus. Two Mongolian H7 viruses belonged to the Eurasian avian lineage, and they showed 93.2% identity with themselves. The H7 HA genes of Wbf/Mongolia/1/51/07 and Wild duck/Mongolia/1-241/08 were identical to Korean isolate Duck/Kr/BC10/07 (100% identity) and shared 99.0% identity with Wild duck/Kr/SH 20-27/08 (Figure 2b).

Phylogenetic analysis of the 11 N8 genes isolated from Mongolian wild birds identified 2 major separate lineages. Most of the Mongolian isolates and partial Korean viruses belonged to the Eurasian avian lineage, whereas 1 Mongolian isolate (Whooper swan/Mongolia/1-14/07) clustered in the American avian lineage with Korean viruses in poultry and wild birds (Figure 2c).

Phylogenetic Analysis of the Internal Genes

Phylogenetic dendrograms of the internal genes were compared with those of Mongolian isolates in wild birds to assess their relationship with the H5N1 HPAI viruses that caused outbreaks from 2003 to 2008, as well as low-pathogenic avian influenza (LPAI) viruses in domestic poultry in Korea (Figure A1). The polymerase basic protein 2 (PB2) and polymerase basic protein 1 (PB1) genes in Mongolian isolates belonged to the Eurasian lineage, which were clustered differently from the H5N1 HPAI viruses of the Gs/GD-like lineage (Figure A1a and A1b). Likewise, the nucleoprotein (NP) and matrix (M) genes showed a similar pattern with the PB2 and PB1 genes (Figure A1c and A1d). However, the polymerase acidic protein (PA) genes, unlike the aforementioned internal genes, did not display a separate sublineage with the goose/Guangdong/1/1996 (Gs/GD)-like lineage, although these PA genes showed high homology with the Eurasian lineage; they were closely related to the clade 2.5, clade 2.2, and clade 2.3.2 H5N1 HPAI viruses, which are endemic and have been circulating in regions of Eurasia, including China, Japan, Mongolia, and Korea (Figure A1e). The nonstructural genes comprised 2 major sublineages: 12 viruses clustered to allele B, whereas 9 of the nonstructural genes clustered to a different allele (allele A), which was separated from the H5N1 HPAI virus of the Gs/GD-like lineage (Figure A1f). It is interesting that the low-pathogenic H7 virus from Duck/Kr/BC10/07 was closely related to the Mongolian isolates at all the internal genes.

DISCUSSION

The present study was implemented to monitor wild birds based on the concern that wild birds could disseminate AIV between Mongolia and Korea by virtue of a shared flyway. The National Veterinary Research and Quarantine Service in Korea and the State Central Veterinary Laboratory in Mongolia collaboratively began a heightened AIV field surveillance. From 2007 to 2009, 1,528 fecal samples were collected from major habitats and outbreak sites of H5N1 HPAI in wild birds in Mongolia; 21 LPAI viruses, including 2 H7 subtypes, were isolated. Two cases of outbreaks of H5N1 HPAI in dead or moribund birds in Mongolia that occurred in May and August 2009 during this study were reported previously (Kang et al., 2011).

In this study, the prevalence of AIV in wild birds in Mongolia was relatively low (1.37%) and was high only in the southeastern region, in Sukhbaatar province. This low prevalence is consistent with a study that reported isolation of 9 viruses from 533 samples (1.7%) from wild birds in Mongolia (Spackman et al., 2009). As shown in Table 2, one of the most frequently isolated subtypes in our study was H3 (61.9%), and the most prevalent HA/NA subtype was H3N8. This result agreed in part with the subtypes reported in other studies (Krauss et al., 2004; Olsen et al., 2006; Bui et al., 2011). In previous studies conducted in Korea, the H3 subtype was isolated at very diverse rates (4.0 to 45.1%) in wild birds and poultry (Song et al., 2008; Kang et al., 2010; Lee et al., 2010b). Because the present study was conducted for only 3 yr and the numbers of AIV isolates were small, the data were insufficient to establish trends in Mongolia. Further work is needed to illuminate the prevalence of AIV and its subtypes.

We exploited an established bar-coding system using mitochondrial DNA to identify virus recovered from fecal samples. Analysis of these data revealed that Anseriformes, such as the whooper swan, pintail, and ruddy shelduck, were the only orders that shed AIV. This result is consistent with an earlier report that Anseriformes are the major order of H5- and H7-shedding viruses in wild birds in Korea (Kang et al., 2010). In the future, the bar-coding system will be applied to all fecal samples; this will broaden the data obtained while avoiding the onerous sampling from captured birds.

Phylogenetic analysis for the surface genes of the Mongolian isolates was performed to assess their genetic relationships with those of domestic poultry and wild birds in Korea. The HA gene of H3 viruses isolated from wild birds in Mongolia clustered together in a group with Korean isolates and showed genetic diversity. Among these isolates, the H3 genes of domestic chickens and ducks in Korea were distinguishable from those of wild birds in both countries (Figure 2a). This result implies that the H3 viruses of poultry in Korea might have originated from wild birds and might have become established as genetically stable forms.

Two LPAI H7 viruses isolated in this study belonged to the Eurasian avian lineage and were closely related to those of wild birds in Korea. However, among the HA gene of the H7 viruses, there was little genetic diversity between wild birds and poultry in both countries, unlike the H3 viruses. This result presumes that H7 viruses originating from wild birds might not have become established in poultry because of eradication strategies for the subtypes H5 and H7 in poultry of Korea.

The N8 genes of Mongolian and Korean isolates appear to have evolved into 2 major lineages. Most of them clustered phylogenetically with viruses isolated in Eurasia, whereas one of the Mongolian viruses [Whooper swan/Mongolia/1/14/07 (H3N8)] and some Korean viruses in 1 chicken and in wild birds belonged to the North American lineage (Figure 2c). Previously, an AIV outbreak caused by H9N8 was first detected in chickens on Korean native chicken farms; this isolate was determined to contain the N8 gene of North American lineage circulating in a Korean wild bird population (Kwon et al., 2006). Manzoor et al. (2008) suggested that some ducks, such as the Northern pintail (A. acuta), may serve as potential intercontinental carriers of influenza viruses in Japan. This finding indicates that through overlapping flyways, AIV have become mixed among different migratory bird species, driving the spread of the virus over long distances within Asia and between continents (Wang et al., 2008).

Phylogenetic analysis of the internal genes revealed that the PB2, PB1, NP, M, and NS genes clustered separately from the H5N1 HPAI viruses of the Gs/GD-like virus, whereas the PA genes were not distinguishable from this lineage and showed multiple genotypes (Figure A1a to A1f). These relationships suggest that the multiple gene pools of wild birds provide Gs/GD-like viruses with their genes in part by reassortment events. Pu et al. (2009) also reported that the PB2 and PA genes of the H3 viruses possessed the highest similarities with H5N1 HPAI viruses circulating in northern China.

In conclusion, the results suggest that the presently obtained Mongolian AIV isolates have evolved with genetically multiple genotypes and are closely related to those of AIV in poultry as well as in wild birds of Korea. Therefore, LPAI viruses as well as HPAI viruses in both countries should be monitored closely to understand their epidemiological relationships, and further surveillance should continue through the international cooperation with countries sharing common migratory flyways.

APPENDIX

Figure A1

Phylogenetic trees of the inner genes: (a) PB2, (b) PB1, (c) NP, (d) M, (e) PA, and (f) NS. The Mongolian viruses isolated in our study are denoted in boldface, and Korean viruses isolated in poultry and wild birds or available in GenBank (http://www.ncbi.nlm.nih.gov/genbank/) are italicized. The nucleotide sequences were analyzed using Clustal X (version 1.83; KVL Bioinformatics, Copenhagen, Denmark), and phylogenetic trees were constructed by the neighbor-joining method. The robustness of groupings was assessed by bootstrap resampling of 1,000 replicate trees.

Figure A1

Phylogenetic trees of the inner genes: (a) PB2, (b) PB1, (c) NP, (d) M, (e) PA, and (f) NS. The Mongolian viruses isolated in our study are denoted in boldface, and Korean viruses isolated in poultry and wild birds or available in GenBank (http://www.ncbi.nlm.nih.gov/genbank/) are italicized. The nucleotide sequences were analyzed using Clustal X (version 1.83; KVL Bioinformatics, Copenhagen, Denmark), and phylogenetic trees were constructed by the neighbor-joining method. The robustness of groupings was assessed by bootstrap resampling of 1,000 replicate trees.

ACKNOWLEDGMENTS

This work was supported by a grant from the National Veterinary Research and Quarantine Service (No. M-AD15-2006-09-01), Anyangro, Republic of Korea.

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